1. Field of the Invention
The present invention relates to a microscope examination method, an optical stimulation apparatus, and a microscope examination apparatus employed in optical marking. Optical marking utilizes a substance that produces fluorescence in response to an optical stimulus, such as a fluorescent protein or a caged compound, or that activates another fluorescent substance.
This application is based on Japanese Patent Application No. 2004-152994, the content of which is incorporated herein by reference.
2. Description of Related Art
A conventionally known microscope examination apparatus including this type of optical stimulation apparatus is the microscope examination apparatus disclosed, for example, in Atsushi Miyawaki, et al., “Special Review, Optical techniques using the new fluorescent protein kaede”, Cell Technology, Vol. 22, No. 3, 2003, pp 316-326 (hereinafter referred to as reference 1).
This microscope examination apparatus is an inverted-type incident-light fluorescence microscope having an observation light source formed of a xenon lamp and a fluorescence filter for carrying out fluoroscopy. In addition, this microscope is also provided with a xenon lamp, an excitation filter, and a field stop for forming a spot of ultraviolet light serving as an optical stimulus. The optical stimulus is made incident on the specimen along the same optical axis by means of a dichroic mirror disposed in the light path of the observation light source.
With this microscope examination apparatus, the optical system, including the field stop, the focusing lens, the objective lens, and so forth, can be precisely adjusted, thus making it possible to precisely position the spot of ultraviolet light at the center of the field of view used for fluoroscopy. Therefore, the location in the object under examination, such as a cell, where the optical stimulus is to be applied can be aligned with the center of the field of view and irradiated with the spot of ultraviolet light. Thus, the optical stimulus can be accurately applied to the target cell, which allows optical marking to be carried out.
In such a case, the optical stimulus location in the object under examination is restricted to a single point at the center of the field of view in the microscope apparatus in reference 1. Therefore, when an optical stimulus is to be accurately applied to that location, it is essential to shift the relative positional relationship in directions orthogonal to the optical axis of the objective lens and the object under examination.
When examining cells and so forth, in order to maintain the viability of the cells, it is customary to carry out examination of the cells while they are disposed in a predetermined amount of liquid, such as a culture medium or the like. However, one drawback with this technique is that moving the object under examination while keeping the objective lens fixed causes the cells to move around in the liquid, thus changing the examination conditions. Also, when moving the objective lens while keeping the object under examination fixed, it is necessary to move the entire optical system including the objective lens. In order to move it with accuracy, the apparatus inevitably becomes larger and the cost is also increased. This is another drawback.
Another possible method is that in which a field stop is moved in directions orthogonal to the optical axis. This method does not suffer from the drawbacks mentioned above. However, the irradiation position of the spot of ultraviolet light is arbitrarily moved in the optical field, which differs from the methods described above in which the spot is fixed at the center of the field of view. Therefore, this method suffers from a problem in that it is difficult to accurately specify the irradiation position.